Fanconi anemia (FA) is an autosomal recessive disorder characterized clinically by a progressive pancytopenia; diverse developmental abnormalities most commonly affecting upper limbs, skin, and kidneys; and a predisposition to malignancy. Spontaneous chromosomal breakage and hypersensitivity to a variety of clastogenic agents are cellular features of the syndrome. The molecular basis for the disorder is unknown. We plan to study genetic heterogeneity in FA by complementation analysis in euploid somatic cell hybrids. Lymphoblastoid cell lines (B cell lines transformed by Epstein-Barr virus) will be derived from well charactgerized FA patients. HGPRT-deficient variant lines will be developed from these by mutagnizing the cells with ethyl methanesulfonate, and selecting for 8-azaguanine-resistant clones. These mutant FA cells will be fused with peripheral blood lymphocytes from different FA patients. Hybrid cells will be selected in hypoxanthine-aminopterin-thymidine (HAT) medium. A decrease in the extraordinary sensitivity of FA cells to the clastogenic effect of diepoxybutane (DEB) will be evidence for genetic complementation. Hybrids will also be constructed between FA homozygous, FA heterozygous and normal cells in various combinations. These experiments will enable us to study gene dosage effects and may be useful for heterozygote detection. Laboratory studies in selected patients will deal with questions relating to the molecular defect in FA as reflected in the differential sensitivity of cells to the clastogenic effect of a diverse group of genotoxic agents, including mutagenic and carcinogenic chemicals, tumor-promoters, and oxygen. The sensitivity of FA heterozygote cells cultured at various oxygen tensions to a variety of DNA-damaging agents will also be studied for use as a method for heterozygote detection. The comprehensive approach to the study of FA outlined in this proposal may provide information that will aid in the identification of the genetic defect in FA, as well as further our understanding of aplastic anemia in general.